This application claims priority to co-pending United Kingdom Patent Application No. GB 0022844.5, filed Sep. 18, 2000 and co-pending United Kingdom Patent Application No. GB 0029920.6, filed Dec. 7, 2000, the entire contents of each are incorporated herein by reference in their entirety.
The present invention relates to methods for prevention and treatment of bone-related or nutrition-related disorders using a GLP molecule or GLP activator either alone or in combination with another therapeutic. The present invention also encompasses methods of diagnosing or monitoring the progression of a disorder. The invention also encompasses methods of monitoring the effectiveness of treatment of the invention.
Glucagon and Related Peptides
Glucagon is a hormone that is released in response to low glucose levels and stimulates glucose production. Thus, it plays a role in counteracting insulin in blood glucose homeostasis (Unger and Orci, 1990, Glucagon in Diabetes Mellitus, 4th edition, Elsevier p. 104-120). Glucagon arises from the post-translational processing of a larger precursor molecule, proglucagon.
Proglucagon is produced in both the xcex1-cells of the pancreas as well as in the enteroendocrine L-cells of the intestine. It is subject to differential processing in the different tissues in which it is expressed. For example, glucagon is selectively excised from the precursor in the pancreas while two smaller peptides, glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2), are produced in the intestine. GLP-1 and GLP-2 consist of amino acid residues 78-107 and 126-158 of proglucagon respectively (Bell et al., 1983, Nature 304: 368-371; Buhl et al., 1988, J. Biol. Chem., 263:8621; Nishi and Steiner, 1990, Mol. Endocrinol. 4:1192-1198; Irwin and Wong, 1995, Mol. Endocrinol. 9:267-277).
Glucagon and GLP-1 have competing biological activities. GLP-1 stimulates insulin secretion, glucose uptake, and cAMP formation in response to the presence and absorption of nutrients in the gut, whereas glucagon increases glucose output by the liver, skeletal muscle tissue, and adipose tissue during periods of fasting (see, e.g., Mojsov, 1992, Int. J. Pep. Prot. Res. 40:333-343; Andreasen et al., 1994, Digestion 55:221-228). Specific GLP-1 receptors have been identified (Thorens, 1992, Proc. Natl. Acad. Sci. 89:8641-8645) which are distinct from the glucagon receptor (Jelinek et al., 1993, Science 259:1614-1616).
GLP-2 is 33 amino acid fragment of proglucagon. Various vertebrate forms (including human) of GLP-2 have been reported. GLP-2 has intestinotrophic activity (U.S. Pat. No. 5,834,428).
When administered exogenously, GLP-2 can produce a marked increase in the proliferation of small intestinal epithelium in mice, with no apparent side effects (Drucker et al., 1996, Proc. Natl. Acad. Sci. 93:7911-7916). Moreover, GLP-2 increases maximal transport rate of D-glucose across the intestinal basolateral membrane (Cheeseman and Tseng, 1996, Am. J. Phys. 271:G477-G482). GLP-2 may act via a G-protein-coupled receptor (Munroe et al., 1999, Proc. Natl. Acad. Sci. 96:1569-1573).
Disorders
Obesity is one of the most common medical disorders, affecting about 40% of the American population. Mortality from obesity in the United States is estimated at 300,000 to 400,000 per year. Although the etiology of obesity is not fully understood, obesity occurs when energy intake exceeds energy expenditure. Hypothalamic structures, which have complex interconnections with the limbic system and other brain structures, control appetite. In addition, the amount and distribution of a person""s body fat may be genetically predetermined and influenced by hormones. Among the agents known to be involved in appetite control are leptin, GLP-1, GLP-2, and neuropeptide-Y.
Osteoporosis is the most common form of metabolic bone disease. It affects more than 25 million people in the United States and causes more than 1.3 million bone fractures each year, including approximately 500,000 spine, 250,000 hip and 240,000 wrist fractures. Hip fractures are the most serious consequence of osteoporosis, with 5-20% of patients dying within one year of the fiacture and over 50% of survivors being incapacitated.
Osteoporosis is commonly observed in post-menopausal women, but it also occurs in elderly and young individuals. The disease is characterized by low bone mass and a deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. Although the etiology of osteoporosis is not known, its onset is associated with several factors such as increased age, decreased hormone level, and decreased calcium levels. Osteoporosis may occur in elderly men as androgen levels fall. Androgens play an important role in bone formation/maintenance and promote the synthesis of collagen, which provides a repository for the calcium and phosphorus. Osteoporosis may also be due to increased secretion of parathyroid hormone, which reduces bone formation and enhances bone absorption. Osteoporosis can also be caused by kidney degeneration, which reduces the activity of hydroxylase-activating vitamin D, decreasing intestinal calcium absorption, and precipitating the loss of bone matrix. Mobilization of nutrient stores in bone can be achieved by stimulating osteoclastic bone resorption. Likewise, resorptive activity can be reversed by increasing dietary availability of nutrients.
Dietary intake of calcium has been shown to regulate bone metabolism. Intake of oral glucose has recently been shown to decrease bone resorption, resulting in a fully expressed decrease within two hours following glucose administration (GB Patent Application No. 0007492.2). This response to glucose intake is independent of gender and age. A comparable effect was also demonstrated following protein administration (unpublished communication).
Bone-related disorders are characterized by bone loss resulting from an imbalance between bone resorption and bone formation. The potential for bone loss is directly related to the bone""s normal rate of resorption and can amount to over 5% per year in humans immediately following menopause.
There are currently two main types of pharmaceutical treatment for osteoporosis, both aimed at reduction of bone resorption. The first involves the administration of an anti-resorptive compound. For example, estrogen has been used as an anti-resorptive agent to reduce fractures. However, estrogen fails to restore bone to levels of that in a skeleton of a young adult. Furthermore, long-term estrogen therapy has been implicated in a variety of disorders, including an increase in the risk of uterine cancer, endometrial cancer, and possibly breast cancer (Persson et al., 1997, xe2x80x9cHormone replacement therapy and the risk of breast cancer. Nested case-control study in a cohort of Swedish women attending mammography screeningxe2x80x9d, Int. J. Can. 72:758-761). For these reasons, many women avoid treatment of osteoporosis with estrogen.
A second type of pharmaceutical therapy for treating osteoporosis uses an agent that inhibits bone resorption as well as promotes bone formation and increases bone mass. These agents, such as alendronate, typically restore the amount of bone to that of an established premenopausal skeleton. However, alendronate administration can cause undesirable side effects, for example, or gastric ulceration (Graham et al., 1999, Aliment Pharmacol. Ther. 4:515-9).
The significant risks associated with the currently available pharmaceutical therapies (such as estrogen and alendronate) highlight the need to develop safer therapies for treating or preventing osteoporosis and other bone-related disorders. Therefore, there is a need for methods for treating or preventing a bone disorder, such as osteoporosis, that do not carry the aforementioned risks.
The present invention relates to the prevention or treatment of a bone-related or nutrition-related disorder comprising administering to a patient a composition that increases GLP-2 activity. Compositions of the invention comprise a GLP-2 molecule or a GLP-2 activator. One or more additional therapeutic agents can be administered in conjunction with the compositions of the invention.
Also contemplated by the invention are methods for diagnosing a bone-related or nutrition-related disorders in a patient comprising:
(a) determining the level of GLP-2 molecule expressed in a normal tissue and a test tissue;
(b) comparing said levels of GLP-2 molecule expression in said tissues, wherein a decrease said level of GLP-2 molecule expression in said test tissue indicates a bone-related or nutrition-related disorder.
Also contemplated by the invention are methods of monitoring the progression of a bone-related or nutrition-related disorder in a patient comprising:
(a) determining the level of GLP-2 molecule expressed in a first diseased tissue;
(b) determining the level of GLP-2 molecule expressed in a second diseased tissue, wherein said second diseased tissue is taken from the same patient as said first diseased tissue but at a later date; and
(c) comparing said levels of GLP-2 molecule expression in said first and second diseased tissues, wherein a decrease said level of GLP-2 molecule expression in said second diseased tissue indicates progression of said bone-related or nutrition-related disorder.
Also contemplated by the invention are methods of determining the effectiveness of treatment with a GLP molecule or GLP activator in a patient comprising:
(a) determining the level of one or more markers of bone resorption from a first patient tissue samples prior to said treatment and a second patient tissue sample after said treatment;
(b) comparing said levels of one or more markers in said tissue samples, wherein a decrease in said level in said second tissue sample indicates effective treatment.
Also contemplated by the invention are methods of determining the effectiveness of treatment with a GLP molecule or GLP activator in a patient comprising:
(a) determining the level of one or more markers of nutrition-related disorder from a first patient tissue samples prior to said treatment and a second patient tissue sample after said treatment;
(b) comparing said levels of one or more markers in said tissue samples, wherein a modification in said level in said second tissue sample indicates effective treatment
Definitions
As used herein, the phrase xe2x80x9cGLPxe2x80x9d refers to GLP-1 or GLP-2.
As used herein, the phrase xe2x80x9cGLP moleculesxe2x80x9d refers to GLP peptides, fragments of GLP peptides, nucleic acids that encode GLP peptides or fragments, or variants thereof.
As used herein, the term xe2x80x9cvariantxe2x80x9d or xe2x80x9cvariantsxe2x80x9d refers to variations of the nucleic acid or amino acid sequence of GLP molecules. Homologues and analogs of a GLP molecule of the invention are contemplated. Encompassed within the term xe2x80x9cvariant(s)xe2x80x9d are nucleotide and amino acid substitutions, additions, or deletions of GLP-1 or GLP-2 molecules. Also encompassed within the term xe2x80x9cvariant(s)xe2x80x9d are chemically modified natural and synthetic GLP-1 or GLP-2 molecules.
As used herein, the term xe2x80x9canalogxe2x80x9d or xe2x80x9canalogsxe2x80x9d as used herein refers to a polypeptide that possesses a similar or identical function to a GLP polypeptide or a fragment of a GLP polypeptide, but does not necessarily comprise a similar or identical amino acid sequence of a GLP polypeptide or a fragment of a GLP polypeptide, or possess a similar or identical structure of a GLP polypeptide or a fragment of a GLP polypeptide. A polypeptide that has a similar amino acid sequence refers to a polypeptide that satisfies at least one of the following: (a) a polypeptide having an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence of a GLP polypeptide or a fragment of a GLP polypeptide described herein; (b) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding a GLP polypeptide or a fragment of a GLP polypeptide described herein of at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, or at least 30 amino acid residues; and (c) a polypeptide encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the nucleotide sequence encoding a GLP polypeptide or a fragment of a GLP polypeptide described herein. A polypeptide with similar structure to a GLP polypeptide or a fragment of a GLP polypeptide described herein refers to a polypeptide that has a similar secondary, tertiary or quaternary structure of a GLP polypeptide or a fragment of a GLP polypeptide described herein. The structure of a polypeptide can determined using methods known to those skilled in the art, including but not limited to, X-ray crystallography, nuclear magnetic resonance, and crystallographic electron microscopy.
To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nuclcotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions/total number of positionsxc3x97100%). In one embodiment, the two sequences are the same length.
The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present invention. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score-50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (e.g., http://www.ncbi.nlm.nih.gov). Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
As used herein, the term xe2x80x9cfragmentxe2x80x9d or xe2x80x9cfragmentsxe2x80x9d as used herein refers to a peptide or polypeptide having an amino acid sequence of at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, or at least 30 contiguous amino acid residues of the amino acid sequence of a GLP polypeptide.
As used herein, the phrase xe2x80x9cGLP activatorxe2x80x9d or xe2x80x9cGLP activatorsxe2x80x9d refers to any molecule or compound that increases the activity of GLP in a patient. The invention encompasses, e.g., GLP agonists, GLP receptor agonists, agonist of the GLP signal transduction cascade, compounds that stimulate the synthesis or expression of endogenous GLP, compounds that stimulate release of endogenous GLP, and compounds that inhibit inhibitors of GLP activity (ie., an inhibitor of a GLP antagonist).
As used herein, the term xe2x80x9cpatientxe2x80x9d is an animal, such as, but not limited to, a cow, monkey, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit, and guinea pig, and is more preferably a mammal, and most preferably a human.
As used herein, the phrase xe2x80x9ctherapyxe2x80x9d or xe2x80x9ctherapeutic agentxe2x80x9d refers to any molecule, compound, or treatment that assists in the treatment of a disease, especially a bone-related disorder and a nutrition-related disorder. As such, therapy includes, but is not limited to, radiation therapy, chemotherapy, dietary therapy, physical therapy, and psychological therapy.
As used herein, the phrase xe2x80x9cbone-related disorderxe2x80x9d refers to a disorder wherein bone formation, deposition, or resorption is abnormal. Bone-related disorders include, but are not limited to, osteoporosis, hypercalcemia of malignancy, osteopenia due to bone metastases, periodontal disease, hyperparathyroidism, periarticular erosions in rheumatoid arthritis, Paget""s disease, osteodystrophy, myositis ossificans, Bechterew""s disease, malignant hypercalcernia, osteolytic lesions produced by bone metastasis, bone loss due to immobilization, bone loss due to sex steroid hormone deficiency, bone abnormalities due to steroid hormone treatment, bone abnormalities caused by cancer therapeutics, osteomalacia, Bechet""s disease, osteomalacia, hyperostosis, osteopetrosis, metastatic bone disease, immobilization-induced osteopenia, and glucocorticoid-induced osteoporosis.
As used herein, the phrase xe2x80x9cnutrition-related disorderxe2x80x9d refers to a disorder characterized by an abnormal level of food intake or body weight gain/loss and complications from such disorders. Nutrition-related disorders include, but are not limited to, obesity, anorexia, cachexia, bulimia, and other wasting diseases characterized by loss of appetite, diminished food intake, or body weight loss. Complications include, but are not limited to, insulin resistance, diabetes mellitus, hypertension, cardiovascular disease, pseudotumor, cerebri, hyperlipidemia, sleep apnea, cancer, pulmonary hypertension, cardiovascular disease, cholecystitis, and osteoarthritis.
As used herein, the phrase xe2x80x9cpharmaceutically acceptablexe2x80x9d refers to an agent that does not interfere with the effectiveness of the biological activity of an active ingredient, and which may be approved by a regulatory agency of the Federal government or a state government, or is listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly for use in humans. Accordingly, suitable pharmaceutically acceptable carriers include agents that do not interfere with the effectiveness of a pharmaceutical composition.
As used herein, the phrase xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d refers to salts prepared from pharmaceutically acceptable, preferably nontoxic, acids and bases, including inorganic and organic acids and bases, including but not limited to, sulfuric, citric, maleic, acetic, oxalic, hydrochloride, hydro bromide, hydro iodide, nitrate, sulfate, bisulfite, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e., 1,1xe2x80x2-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Pharmaceutically acceptable salts include those formed with free amino groups such as, but not limited to, those derived from hydrochloric, phosphoric, acetic, oxalic, and tartaric acids. Pharmaceutically acceptable salts also include those formed with free carboxyl groups such as, but not limited to, those derived from sodium, potassium, ammonium, sodium lithium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, and procaine.
As used herein, the term xe2x80x9ccarrierxe2x80x9d refers to a diluent, adjuvant, excipient, or vehicle. Such carriers can be sterile liquids, such as saline solutions in water, or oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
As used herein, the tern xe2x80x9cmineralxe2x80x9d refers to a substance, preferably a natural substance, that contain calcium, magnesium or phosphorus. Illustrative nutrients and minerals include beef bone, fish bone, calcium phosphate, egg shells, sea shells, oyster shells, calcium carbonate, calcium chloride, calcium lactate, calcium gluconate and calcium citrate.
As used herein, the term xe2x80x9cbiological samplexe2x80x9d is broadly defined to include any cell, tissue, organ or multicellular organism. A biological sample can be derived, for example, from cells or tissue cultures in vitro. Alternatively, a biological sample can be derived from a living organism or from a population of single cell organisms. Preferably, the biological sample is live tissue. More preferably, the biological sample is live bone or adipose tissue.
As used herein, the term xe2x80x9cGIPxe2x80x9d refers to glucose-dependent insulinotropic polypeptide. GIP is an incretin that stimulates insulin secretion directly in a glucose-dependent manner.
As used herein, the term xe2x80x9cS-CTXxe2x80x9d refers to a serum C-telopeptide fragment of collagen type I degradation.
As used herein, the phrase xe2x80x9cisolated polypeptide or peptidexe2x80x9d refers to a polypeptide or peptide that is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language xe2x80x9csubstantially free of cellular materialxe2x80x9d includes preparations of protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a xe2x80x9ccontaminating proteinxe2x80x9d). When the protein, peptide, or fragment thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. When the protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly such preparations of the protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest. In preferred embodiments, purified or isolated preparations will lack any contaminating proteins from the same animal from which the protein is normally produced, as can be accomplished by recombinant expression of, for example, a human protein in a non-human cell.
As used herein, the phrase xe2x80x9cisolated nucleic acid moleculexe2x80x9d refers to a nucleic acid molecule which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Preferably, an isolated nucleic acid molecule is free of sequences (preferably protein encoding sequences) which naturally flank the nucleic acid (ie., sequences located at the 5xe2x80x2 and 3xe2x80x2 ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. In other embodiments, the isolated nucleic acid is free of intron sequences. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kB, 4 kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an isolated nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, the nucleic acid molecules of the invention comprise a contiguous open reading frame encoding a polypeptide of the invention.
As used herein, the phrase xe2x80x9chybridizes under stringent conditionsxe2x80x9d is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (65%, 70%, 75%, 80%, or preferably 85% or more) identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989), 6.3.1-6.3.6, which describes aqueous and non-aqueous methods, either of which can be used. Another preferred, non-limiting example of stringent hybridization conditions are hybridization in 6xc3x97sodium chloride/sodium citrate (SSC) at about 45xc2x0 C., followed by one or more washes in 2.0xc3x97SSC at 50xc2x0 C. (low stringency) or 0.2xc3x97SSC, 0.1% SDS at 50-65xc2x0 C. (high stringency). Another preferred example of stringent hybridization conditions are hybridization in 6xc3x97sodium chloride/sodium citrate (SSC) at about 45xc2x0 C., followed by one or more washes in 0.2xc3x97SSC, 0.1% SDS at 50xc2x0 C. Another example of stringent hybridization conditions are hybridization in 6xc3x97sodium chloride/sodium citrate (SSC) at about 45xc2x0 C., followed by one or more washes in 0.2xc3x97SSC, 0.1% SDS at 55xc2x0 C. A further example of stringent hybridization conditions are hybridization in 6xc3x97sodium chloride/sodium citrate (SSC) at about 45xc2x0 C., followed by one or more washes in 0.2xc3x97SSC, 0. 1% SDS at 60xc2x0 C. Preferably, stringent hybridization conditions are hybridization in 6xc3x97sodium chloride/sodium citrate (SSC) at about 45xc2x0 C., followed by one or more washes in 0.2xc3x97SSC, 0.1% SDS at 65xc2x0 C. Particularly preferred stringency conditions (and the conditions that should be used if the practitioner is uncertain about what conditions should be applied to determine if a molecule is within a hybridization limitation of the invention) are 0.5 M Sodium Phosphate, 7% SDS at 65xc2x0 C., followed by one or more washes at 0.2xc3x97SSC, 1% SDS at 65xc2x0 C. In one embodiment, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of the GLP nucleic acid, or a complement thereof, corresponds to a naturally-occurring nucleic acid molecule. As used herein, a xe2x80x9cnaturally occurringxe2x80x9d nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g. encoding a natural protein).